The present invention relates generally to the microbial synthesis of phenylalanine and more particularly to novel DNA sequences encoding polypeptide analogs of the E. coli enzyme, chorismate mutase/prephenate dehydratase. In comparisons to the wild type enzyme, the enzymatic activities of the analogs are more resistant to feedback inhibition by phenylalanine. The analog-encoding DNA sequences are therefore useful in supplementing the enzymatic wherewithal of microorganisms employed in phenylalanine production.
In the microbial production of L-phenylalanine in E. coli numerous metabolic enzymes are involved. Among the most significant of these is a bifunctional enzyme, chorismate mutase/prephenate dehydratase (CMPD), which is involved in both the conversion of chorismate to prephenate and prephenate to phenylpyruvate. CMPD has been determined to be the expression product of the E. coli pheA gene, the nucleotide sequence of which has been reported by Hudson et al., J. Mol. Biol., 180, 1023-1051 (1984).
CMPD has been proposed to function enzymatically in a dimeric form comprising two identical polypeptide products of pheA gene expression. The enzyme is subject to "feedback inhibition" of its activities by the metabolic pathway end product, L-phenylalanine. When phenylalanine levels approach 1.0 mM, for example, there is a dramatic slowdown in prephenate dehydratase activity, probably due to participation of phenylalanine in the reversible formation of enzymatically inactive CMPD polypeptide tetramers. [See, e.g., Baldwin et al., Arch. Biochem. Biophys., 211, 66-75 (1981)] At phenylalanine concentrations of about 1.0 mM, prephenate dehydratase activity is reduced by at least 90 percent.
With the advent of recombinant technologies for the cloning and expression of genes, attempts have been made to augment the endogenous CMPD capacity of E. coli host cells employed in phenylalanine production [Forberg et al., J. Biotech., 7, 319-332 (1988); Choi et al., Biotechnol. Lett., 8, 223-228 (1982); Hwang et al., Appl. Microbiol. Biotechnol., 22, 108-113 (1985); Gil et al., Enzyme Microb. Technol., 7, 370-372 (1985); Park et al., Chem. Eng. Commun., 45, 185-196 (1986)].
Mutant E. coli strains have been reported to produce CMPD enzyme substantially free of phenylalanine feedback inhibition. See, e.g., Tribe, Published Australian Application No. 72727/81.
Backmann et al., U.S. Pat. No. 4,753,883, reports that transformation of host cells with "mutant" DNA sequences encoding CMPD analog polypeptides which are less sensitive to phenylalanine inhibition on the basis that ". . . the catalytically critical segment of E. coli CMPD lies within its N-terminal 337 amino acids, that phenylalanine feedback sensitivity depends on a single amino acid tryptophan 338, and that deletion of the entire 49 C-terminal amino acids does not destroy catalytic activity but does substantially destroy feedback sensitivity". Backmann et al. proposes the development of plasmid vectors incorporating DNA sequences encoding CMPD Trp.sup.338 deletion as well as substitution analogs involving Trp.sup.338 and the use of such plasmid vectors to transform microbial hosts for phenylalanine production.